From the 1970s, it was easier to analyze the DNA molecule with restriction enzyme isolation.
These enzymes are endonucleases, ie inside (hence the prefix endo- inside) DNA molecules by cutting them into well-defined locations.
These are enzymes normally produced by bacteria that have the property of defending them from invading viruses. These substances always "poke" the DNA molecule at certain points, leading to the production of fragments containing adhesive tips, which can bind to other ends of DNA molecules that have been cut with the same enzyme.
In Genetic Engineering, obtaining DNA fragments serves to create, in vitro (in test tube or lab), new molecules, cutting and pasting various pieces of information.
One of the first restriction enzymes to be isolated was EcoRI, produced by the bacteria Escherichia coli. This enzyme only recognizes the sequence GAATTC and always acts between the G and the first A.
The "cut" site, the site of an enzyme, is known as the target site. You may ask why this enzyme does not act on the DNA of the bacteria itself? This is not due to the existence of other protective enzymes that prevent the action of restriction enzymes on the genetic material of the bacteria.
At restriction enzymes recognize and act on specific DNA sequencesby catalyzing the destruction of a phosphodiester bond between two consecutive nucleotides attached to certain bases. The nucleotides between which the enzyme cuts, that is, between which it promotes hydrolysis, are within these same specific recognition sequences (see image).
Each DNA molecule can be composed of multiple repeats of the GAATTC sequence over its entire length. Therefore, upon contact with the EcoRI enzyme, the DNA strand can be cleaved "cut" in several places, generating several pieces of different sizes.
We will see below how these different pieces of cleaved DNA are separated.